This invention relates to a method of treating heparin preparations to isolate a heparin fraction having improved anticoagulant activity and to a method of use of the improved preparation.
Heparin is a mucopolysaccharide composed of amino sugar and uronic acid residues which is obtained from beef, porcine, sheep, whale, or other mammalian tissue by extraction with a solution of potassium acetate, alkaline ammonium sulfate and the like. Commercial heparin preparations are now widely available from many U.S. drug companies and are distributed primarily for use as an intravascular anticoagulant. Recently, heparin has been used clinically as a therapeutic agent for preventing intravascular emboli formation which commonly result in pulmonary embolism and stroke.
Heparin preparations are known to be heterogeneous on a molecular level. Thus, they exhibit a considerable degree of polydispersity in molecular size, variations in the ratio of glucuronic acid to iduronic acid, alterations in the amount of ester and N-sulfation, and differing extents of N-acetylation. Changes in any of these parameters have been correlated only to a very limited extent with heparin's anticoagulant potency. Accordingly, it has been widely assumed that its anticoagulant activity is not traceable to a single specific heparin structure, and in any event, no precise relationship between its structure and function has been forthcoming.
The mechanism of the formation of blood clots is known to be the result of a series of enzymatically catalyzed reactions which ultimately result in the production of fibrin, a plasma insoluble protein from which clots are formed. The precursor to fibrin is fibrinogen, a more complex protein which circulates freely in the cardiovascular system and normally comprises between about 3 and 6 percent of the total plasma protein. Fibrinogen is converted to fibrin by the proteolytic enzyme thrombin which is produced as needed from prothrombin. While it has been known for some time that thrombin promotes clotting by its action on fibrinogen, the mechanism of this control has only recently been elucidated and reported.
It has now been reported that antithrombin, another blood protein, reacts with thrombin to form a complex which is incapable of cleaving fibrinogen. Antithrombin has thus been characterized as a thrombin inhibitor. The antithrombin.sub.III -thrombin reaction normally proceeds at a slow rate. However, it has recently been determined that heparin, if present in the plasma, can rapidly react with antithrombin.sub.III to produce a heparin-antithrombin.sub.III complex which, probably because of steric modification, can rapidly interact with thrombin to neutralize its ability to cleave fibrinogen. Thus, in blood, heparin rapidly reacts with antithrombin.sub.III, the complex produced rapidly inhibits thrombin, the neutralized thrombin is incapable of cleaving fibrinogen to fibrin, and the absence of fibrin prevents coagulation.
Coincidentally with the foregoing discoveries, it had been observed that the anticoagulant properties of heparin are operative only in the presence of a plasma component which had been termed "heparin cofactor". It has now become apparent that the thrombin inhibitor identified as antithrombin.sub.III and the heparin cofactor are one and the same blood protein. Thus, the anticoagulant activity of heparin may be traced to its ability to dramatically accelerate the rate at which the antithrombin.sub.III -heparin cofactor (hereinafter referred to as the cofactor) neutralizes thrombin. Furthermore, it has recently been reported that this cofactor, in the presence of heparin, can neutralize the action of serine proteases of the hemostatic mechanism.